Traditional Chinese medicine composition and use thereof

ABSTRACT

The present disclosure relates to the field of drugs and health foods. Disclosed is a traditional Chinese medicine composition comprising GANODERMA polysaccharide, MORINDAE OFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMA polysaccharide, ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil. Experiments show that the traditional Chinese medicine composition has a scientific formulation, synergistic functions, without toxic and side effects, and not only has nutritional value but also inhibits the growth of tumor. Comparing with individual components, the composition significantly increases the percentages of CD4 +  and CD8 +  cells in lymphocytes of the tumor stroma, enhances body immune function, restores normal immune surveillance function, decreases VEGF and TGF-β1 positive cells in tumor tissue, assists in treating tumor, increases antitumor effects of chemotherapy drugs, improves immune status of body, has the function of restoring normal immune surveillance, preventing and assisting in treating tumor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of Chinese Patent Application No.201710799373.3, filed on Sep. 7, 2017, and the disclosures of which arehereby incorporated by reference.

FIELD

The present disclosure relates to the field of drugs and health foods,specifically to a traditional Chinese medicine composition and usethereof, in particular to a traditional Chinese medicine compositionthat contains various vegetable polysaccharides and vegetable oils, anduse thereof.

BACKGROUND

Tumor is a new organism formed by the proliferation of local tissuecells under the action of various tumorigenic factors. According to thehazard of the tumors to the human body and their growth characteristics,tumors are divided into two types: benign tumors and malignant tumors.Benign tumors, for example lipoma, hemangioma, adenoma, cysts and so on,grow slowly, showing an expansive growth, which have intactpseudocapsule over their surface. There is seldom systemic symptomexcept for local symptoms. Benign tumors do not infiltrate intosurrounding tissues or metastasize to the whole body, and are not easyto relapse after surgical resection, and are less harmful to the body.Malignant tumors, for example bone cancer, esophageal cancer, livercancer, lung cancer, leukemia, osteosarcoma and so on, grow rapidly,often infiltrate into the surrounding tissues and metastasize to thewhole body. There is almost no pseudocapsule over the surface ofmalignant tumors. Pathological examination shows atypical karyokinesismitoses. Except for local symptoms, systemic symptoms are obvious.Patients in the late stage often have cachexia, and have a high relapserate after surgical resection, which are greatly harmful to the body.Tumor is currently at the top of the cause of death spectrum and it is amajor health threat.

The main traditional methods for treating malignant tumors includesurgery, radiation therapy and chemotherapy. Surgical treatment is themost important way to treat malignant tumors, especially for early- andmid-stage malignant tumors, which is listed as the preferred method.After surgical resection, some early-stage tumors can be completelycured, and the patients can survive for a long time. Radiation therapy(radiotherapy) uses radiation to induce changes of DNA, chromosomalaberration or break in tissue cells, and ionization of cellular fluid togenerate chemical free radicals, finally leading to deactivation ofcells or their filial generation, so as to break the cells or inhibitthe growth of tumor. Radiation has adverse effects on normal tissuecells, especially with the increasing amount of light radiation, iteasily damages hematopoietic organs and vascular tissues, and causesleukopenia, thrombocytopenia, changes in skin and mucous membranes,gastrointestinal reactions and so on. Chemical drug therapy(chemotherapy) is also known as anticancer drug treatment. It is mainlyapplicable to the comprehensive therapy of mid- and late-stage cancer.Clinically, chemotherapy has a good effect on chorionic epithelioma,acute lymphoblastic leukemia, malignant lymphoma and so on. For othermalignant tumors, chemotherapy can assist surgery or radiotherapy.However, all of the above three conventional treatment methods havecertain limitations and cause certain painful feelings to the patients,especially radiotherapy and chemotherapy have inhibition effects on thenormal immune system in some degree.

With the development of tumor immunology, people are increasingly awarethat the immune system is critical for the surveillance and clearance oftumors. Immunotherapy is regarded as the fourth type of therapy that isexpected to overcome cancer. In the past, people concerned aboutpositive immunity, that is, the direct killing of tumor cells by immunecells. However, at present, the academic community believes that theimmune negative regulation cells have become new targets for treatingcancer. Immune escape of tumor cells is an important prerequisite forthe development of tumors. The reason why this important biologicalevent occurs is that there is an immune negative regulation network inthe tumor microenvironment, and the immune negative regulatory cells andfactors in this network inhibit the surveillance and clearance of tumorcells by immune effector cells.

SUMMARY

An object of the present disclosure is to provide a traditional Chinesemedicine composition having the function of improving tumormicroenvironment, a method for preparing the same, and use thereof.

In order to achieve the goal of the present disclosure, the followingtechnical solutions are used in the present disclosure.

A traditional Chinese medicine composition, which comprises GANODERMApolysaccharide, MORINDAE OFFICINALIS RADIX polysaccharide, POLYGONATIRHIZOMA polysaccharide, ANGELICAE SINENSIS RADIX oil and MYRISTICAESEMEN oil.

Preferably, in the tradition Chinese medicine composition, the weightratio of GANODERMA polysaccharide, MORINDAE OFFICINALIS RADIXpolysaccharide, POLYGONATI RHIZOMA polysaccharide, ANGELICAE SINENSISRADIX oil and MYRISTICAE SEMEN oil is (1 to 5):(1 to 5):(1 to 5):(1 to5):(1 to 5).

More preferably, in the tradition Chinese medicine composition, theweight ratio of GANODERMA polysaccharide, MORINDAE OFFICINALIS RADIXpolysaccharide, POLYGONATI RHIZOMA polysaccharide, ANGELICAE SINENSISRADIX oil and MYRISTICAE SEMEN oil is (1 to 2):(1 to 2):(1 to 2):(1 to2):(1 to 2).

In some embodiments of the present disclosure, in the tradition Chinesemedicine composition, the weight ratio of GANODERMA polysaccharide,MORINDAE OFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMApolysaccharide, ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil is1:1:1.5:1.5:1.5.

In the traditional Chinese medicine composition of the presentdisclosure, GANODERMA polysaccharide, MORINDAE OFFICINALIS RADIXpolysaccharide, POLYGONATI RHIZOMA polysaccharide, ANGELICAE SINENSISRADIX oil and MYRISTICAE SEMEN oil are either extracted from plants orartificially synthesized.

In the traditional Chinese medicine composition of the presentdisclosure, the method for preparing the GANODERMA polysaccharide,MORINDAE OFFICINALIS RADIX polysaccharide or POLYGONATI RHIZOMApolysaccharide is: respectively pulverizing the GANODERMA, MORINDAEOFFICINALIS RADIX or POLYGONATI RHIZOMA, mixing the GANODERMA, MORINDAEOFFICINALIS RADIX or POLYGONATI RHIZOMA fine powders with water in aweight ratio of 1:(2.5 to 10), heating and stirring for extraction for 2to 4 hours at temperature between 70 and 75° C., collecting the filterresidue, extracting the residue for twice according to thesolid-to-liquid ratio, temperature and duration above, combining theextracts of the three times, concentrating under reduced pressure andcentrifuging to give a clarified concentrate, mixing the clarifiedconcentrate with alcohol of 95% (v/v) in a volume ratio of 1:(7 to 10),holding and precipitating at 4° C., centrifuging and collecting theprecipitation, vacuum freeze-drying, and respectively obtaining theGANODERMA polysaccharide, MORINDAE OFFICINALIS RADIX polysaccharide orPOLYGONATI RHIZOMA polysaccharide.

In the traditional Chinese medicine composition of the presentdisclosure, the method for preparing the ANGELICAE SINENSIS RADIX oilis: mixing the ANGELICAE SINENSIS RADIX fine powder with water which is15 times the weight of the ANGELICAE SINENSIS RADIX fine powder, soakingfor 5 hours, subjecting to steam distillation extraction, during thesteam distillation extraction process, adding sodium chloride which is ⅕times the weight of ANGELICAE SINENSIS RADIX, and extracting for 8 hoursat temperature between 140 and 150° C. and with a vapor flux of 2 L/h.The ANGELICAE SINENSIS RADIX fine powder is preferably 10-mesh filteredANGELICAE SINENSIS RADIX fine powder. The ANGELICAE SINENSIS RADIX oilobtained has a light yellow color, with a special aroma of ANGELICAESINENSIS RADIX and a bitter taste, and a yield from 0.2 to 0.3%, whichis preserved in a refrigerator at −20° C.

In the traditional Chinese medicine composition of the presentdisclosure, the method for preparing the MYRISTICAE SEMEN oil is: addingwater which is 15 times the weight of the MYRISTICAE SEMEN fine powderto the MYRISTICAE SEMEN fine powder and mixing, soaking for 1 hour,subjecting to steam distillation, and extracting for 6 hours attemperature between 130 and 140° C. and with a vapor flux of 2 L/h. TheMYRISTICAE SEMEN fine powder is preferably 20-mesh filtered MYRISTICAESEMEN fine powder. The MYRISTICAE SEMEN oil obtained has a yellow color,with a spicy and intense flavor and a taste of musk, and a yield from 10to 12%, which is preserved in a refrigerator at −20° C.

The traditional Chinese medicine composition of the present disclosureis produced by mixing GANODERMA polysaccharide, MORINDAE OFFICINALISRADIX polysaccharide, POLYGONATI RHIZOMA polysaccharide, ANGELICAESINENSIS RADIX oil and MYRISTICAE SEMEN oil.

It is surprisingly found through experiments that the components of thecomposition of the present invention has a scientific formulation, whichshows significantly superior effects than individual component of thetraditional Chinese medicine composition, and the effects are unexpectedby one of ordinary skill in the art. Cytological experiments show thatcomparing with individual component of the traditional Chinese medicinecomposition, the traditional Chinese medicine composition of the presentdisclosure significantly inhibits tumor growth, increases the percentageof CD4⁺ and CD8⁺ cells in tumor interstitial lymphocytes, and decreasesthe expression rate of VEGF and TGF-β1 positive cells in tumor tissue,so as to restore normal immune function for tumor surveillance, preventimmune escape of tumor cells, enhance the anti-tumor effect ofchemotherapeutic drugs, therefore playing a role of preventing and/orassisting in treating tumor.

Thus, the present disclosure provides use of the traditional Chinesemedicine composition in preparing health foods and/or drugs forinhibiting tumor growth.

The present disclosure further provides use of the traditional Chinesemedicine composition in preparing health foods and/or drugs forincreasing the percentage of CD4⁺ and CD8⁺ cells in lymphocytes of tumorstroma.

The present disclosure further provides use of the traditional Chinesemedicine composition in preparing health foods and/or drugs fordecreasing the expression rate of VEGF and TGF-β1 positive cell in tumortissue.

The present disclosure further provides use of the traditional Chinesemedicine composition in preparing health foods and/or drugs forincreasing immunity function.

The present disclosure further provides use of the traditional Chinesemedicine composition in preparing health foods and/or drugs forrestoring normal immune surveillance function.

Restoring normal immune surveillance function of the body includesinhibiting the growth of tumor, increasing percentage of CD4⁺, CD8⁺cells in lymphocytes of tumor stroma, decreasing expression rate of VEGFand TGF-β1 positive cells in tumor tissue, assisting in treating tumorand improving immune status of the body.

The present disclosure further provides use of the traditional Chinesemedicine in preparing health foods and/or drugs for preventing and/orassisting in treating tumor.

The present disclosure further provides drugs and/or health foods forimproving immune function and preventing and/or assisting in treatingtumor, which comprise the traditional Chinese medicine composition,and/or the traditional Chinese medicine composition obtained by themethod of the present disclosure and a pharmaceutically acceptableexcipient or a food acceptable excipient.

The pharmaceutically acceptable excipient or the food acceptableexcipient includes carrier, adjuvant and/or filler, etc.

In some embodiments of the present disclosure, dosage forms of the drugsor the health foods are capsule, tablet, granule, oral liquid, powder,tea bag and other dosage forms of drugs or health foods.

It can be concluded from the technical solutions above that the presentdisclosure provides a traditional Chinese medicine composition, whichcomprises GANODERMA polysaccharide, MORINDAE OFFICINALIS RADIXpolysaccharide, POLYGONATI RHIZOMA polysaccharide, ANGELICAE SINENSISRADIX oil and MYRISTICAE SEMEN oil. Experiments show that thetraditional Chinese medicine composition has a scientific formulationand each component works well with each other. The composition has notoxic and side effects, which not only has nutritional value but alsoinhibits the growth of tumor. Comparing with individual components,polysaccharide composition, oil composition, and compositions that lackat least one component, the traditional Chinese medicine compositionsignificantly increases the percentage of CD4⁺ and CD8⁺ cells inlymphocytes of the tumor stroma, increases body immune function,recovers normal immune surveillance function, decreases the expressionrate of VEGF and TGF-β1 positive cells in tumor tissue, assists intreating tumor, increases antitumor effects of chemotherapy drugs,improves immune status of body, has the function of restoring normalimmune surveillance, and has functions on preventing and assisting intreating tumors.

DETAILED DESCRIPTION

The present disclosure provides a traditional Chinese medicinecomposition and the application thereof. One of ordinary skill in theart can learn from the contents herein and improve the processparameters appropriately. In particular, it shall be noted that all thesimilar substitutions and modifications are apparent to one of ordinaryskill in the art and are to be considered within the scope of thepresent invention. The method and product of the present invention havebeen described with preferred examples. It is apparent that one of theordinary skill in the art can make change or modify the combination tothe method and product of the present invention without departing fromthe spirit, scope and spirit of the invention, therefore realizing andapplying the techniques of the present invention.

In order to understand the present disclosure further, the technicalsolutions in the embodiments of the present disclosure will be describedclearly and completely herein in conjunction with Examples of thepresent disclosure. Apparently, the described examples are only a partof Examples of the present disclosure, rather than all examples. Basedon Examples in the present disclosure, all of other examples, made byone of ordinary skill in the art without any creative efforts, fall intothe protection scope of the present disclosure.

Without special illustration, all the reagents in Examples of thepresent disclosure are commercial products, which can be purchased onthe market.

Example 1 Preparation of GANODERMA Polysaccharide

GANODERMA polysaccharide of the present disclosure was prepared by thefollowing method. GANODERMA was pulverized, and water was added at aweight ratio of GANODERMA:water=1:10. Extraction was performed for 2hours under conditions of heating and stirring at 70° C. The filterresidue was collected and continuously subjected to extraction for twiceaccording to the solid-to-liquid ratio, temperature and duration above.Extracts of the three extractions were combined, concentrated underreduced pressure and centrifuged to obtain a clarified concentrate. 95%alcohol which was 7 times the volume of the concentrate was added to theclarified concentrate, mixed, and the mixture was held for precipitationover night at 4° C. The mixture was centrifuged to collect theprecipitates, and the precipitates were subjected to vacuumfreeze-drying to obtain the GANODERMA polysaccharide.

Example 2 Preparation of MORINDAE OFFICINALIS RADIX Polysaccharide

MORINDAE OFFICINALIS RADIX polysaccharide of the present disclosure wasprepared by the following method. MORINDAE OFFICINALIS RADIX waspulverized, and water was added at a weight ratio of MORINDAEOFFICINALIS RADIX:water=1:5. Extraction was performed for 3 hours underconditions of heating and stirring at 75° C. The filter residue wascollected and continuously subjected to extraction for twice accordingto the solid-to-liquid ratio, temperature and duration above. Extractsof the three extractions were combined, concentrated under reducedpressure and centrifuged to obtain a clarified concentrate. 95% alcoholwhich was 9 times the volume of the concentrate was added to theclarified concentrate, mixed, and the mixture was held for precipitationover night at 4° C. The mixture was centrifuged to collect theprecipitates, and the precipitates were subjected to vacuumfreeze-drying to obtain the MORINDAE OFFICINALIS RADIX polysaccharide.

Example 3 Preparation of POLYGONATI RHIZOMA Polysaccharide

POLYGONATI RHIZOMA polysaccharide of the present disclosure was preparedby the following method. POLYGONATI RHIZOMA was pulverized, and waterwas added at a weight ratio of POLYGONATI RHIZOMA:water=1:5. Extractionwas performed for 3 hours under conditions of heating and stirring at75° C. The filter residue was collected and continuously subjected toextraction for twice according to the solid-to-liquid ratio, temperatureand duration above. Extracts of the three extractions were combined,concentrated under reduced pressure and centrifuged to obtain aclarified concentrate. 95% alcohol which was 9 times the volume of theconcentrate was added to the clarified concentrate, mixed, and themixture was held for precipitation over night at 4° C. The mixture wascentrifuged to collect the precipitates, and the precipitates weresubjected to vacuum freeze-drying to obtain the POLYGONATI RHIZOMApolysaccharide.

Example 4 Preparation of ANGELICAE SINENSIS RADIX Oil

ANGELICAE SINENSIS RADIX oil of the present disclosure was prepared bythe following method. 400 g of ANGELICAE SINENSIS RADIX fine powder (10meshes) was weighted, and water which was 15 times the weight of thefine powder was added and mixed to reach a solid-to-liquid ratio of1:15. The fine powder was soaked for 5 hours and then subjected to steamdistillation extraction. Sodium chloride (80 g) which was ⅕ time theweight of ANGELICAE SINENSIS RADIX was added during the extraction. Theextraction temperature was from 140 to 150° C., the vapor flux was 2L/h, and the duration was 8 hours. The ANGELICAE SINENSIS RADIX oilobtained has a light yellow color, with a special aroma of ANGELICAESINENSIS RADIX and a bitter taste, and a yield from 0.2 to 0.3%, whichwas preserved in a refrigerator at −20° C.

Example 5 Preparation of MYRISTICAE SEMEN Oil

MYRISTICAE SEMEN oil of the present disclosure was prepared by thefollowing method. 400 g of MYRISTICAE SEMEN fine powder (20 meshes) wasweighted, and water which was 15 times the weight of the fine powder wasadded and mixed to reach a solid-to-liquid ratio of 1:15. The finepowder was soaked for 1 hour and then subjected to steam distillationextraction. The extraction temperature was from 130 to 140° C., thevapor flux was 2 L/h, and the duration was 6 hours. The MYRISTICAE SEMENoil obtained has a yellow color, with a spicy and intense flavor and ataste of musk, and a yield from 10 to 12%, which was preserved in arefrigerator at −20° C.

Example 6 Preparation of the Traditional Chinese Medicine Composition

GANODERMA polysaccharide prepared in Example 1, MORINDAE OFFICINALISRADIX polysaccharide prepared in Example 2, POLYGONATI RHIZOMApolysaccharide prepared in Example 3, ANGELICAE SINENSIS RADIX oilprepared in Example 4 and MYRISTICAE SEMEN oil prepared in Example 5were mixed at a weight ratio of 1:1:1.5:1.5:1.5 to obtain a traditionalChinese medicine composition comprising GANODERMA polysaccharide,MORINDAE OFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMApolysaccharide, ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil.

Example 7 Preparation of the Traditional Chinese Medicine Composition

GANODERMA polysaccharide prepared in Example 1, MORINDAE OFFICINALISRADIX polysaccharide prepared in Example 2, POLYGONATI RHIZOMApolysaccharide prepared in Example 3, ANGELICAE SINENSIS RADIX oilprepared in Example 4 and MYRISTICAE SEMEN oil prepared in Example 5were mixed at a weight ratio of 1:5:4:2:4 to obtain a traditionalChinese medicine composition comprising GANODERMA polysaccharide,MORINDAE OFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMApolysaccharide, ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil.

Example 8 Preparation of the Traditional Chinese Medicine Composition

GANODERMA polysaccharide prepared in Example 1, MORINDAE OFFICINALISRADIX polysaccharide prepared in Example 2, POLYGONATI RHIZOMApolysaccharide prepared in Example 3, ANGELICAE SINENSIS RADIX oilprepared in Example 4 and MYRISTICAE SEMEN oil prepared in Example 5were mixed at a weight ratio of 5:1:1:5:1 to obtain a traditionalChinese medicine composition comprising GANODERMA polysaccharide,MORINDAE OFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMApolysaccharide, ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil.

Example 9 Preparation of the Traditional Chinese Medicine Composition

GANODERMA polysaccharide prepared in Example 1, MORINDAE OFFICINALISRADIX polysaccharide prepared in Example 2, POLYGONATI RHIZOMApolysaccharide prepared in Example 3, ANGELICAE SINENSIS RADIX oilprepared in Example 4 and MYRISTICAE SEMEN oil prepared in Example 5were mixed at a weight ratio of 1:2:1:2:1 to obtain a traditionalChinese medicine composition comprising GANODERMA polysaccharide,MORINDAE OFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMApolysaccharide, ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil.

Example 10 Preparation of the Traditional Chinese Medicine Composition

GANODERMA polysaccharide prepared in Example 1, MORINDAE OFFICINALISRADIX polysaccharide prepared in Example 2, POLYGONATI RHIZOMApolysaccharide prepared in Example 3, ANGELICAE SINENSIS RADIX oilprepared in Example 4 and MYRISTICAE SEMEN oil prepared in Example 5were mixed at a weight ratio of 2:4:5:1:5 to obtain a traditionalChinese medicine composition comprising GANODERMA polysaccharide,MORINDAE OFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMApolysaccharide, ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil.

Example 11 Test the Function of the Traditional Chinese MedicineComposition on Preventing or Assisting in Treating Tumor

GANODERMA polysaccharide, MORINDAE OFFICINALIS RADIX polysaccharide,POLYGONATI RHIZOMA polysaccharide, ANGELICAE SINENSIS RADIX oil,MYRISTICAE SEMEN oil and the traditional Chinese medicine compositionsobtained in examples 1 to 10 above were subjected to cell experiments bythe inventors. The experiments were shown hereinafter.

1. Experiment Materials

1.1 Experiment Equipment and Reagents

Flow cytometer: BD, Calibo.

Analytical balance: Beijing Sartorius Co., Ltd., Sartorius BS124S type.

Electronic balance: Beijing Sartorius Co., Ltd., Sartorius BL1500 type.

Carbon dioxide constant-temperature cell incubator (Nuaire).

Low-temperature high-speed centrifuge (Hunan Xingke TGL-16G).

The CD4⁺, CD8⁺ and antibody for flow cytometry were purchased from BD.

Lymphocyte separation solution (Guanzhou Zhanchen Biological TechnologyCo., Ltd., China)

Fluorescein isothiocyanate labeled anti-mouse CD4 and CD8a monoclonalantibody (BioLegend, Inc., U.S.A.)

VEGF and TGF-β1 immunocytochemistry kit (Beijing Zhongshan BiologicalTechnology Co., Ltd., China)

1.2 Experimental Drugs

The GANODERMA polysaccharide, MORINDAE OFFICINALIS RADIX polysaccharide,POLYGONATI RHIZOMA polysaccharide, ANGELICAE SINENSIS RADIX oil andMYRISTICAE SEMEN oil and the traditional Chinese medicine compositionsprepared in examples 1 to 10, store at 4° C. and avoid light.

1.3 Experimental Animals

Female SPF mice were purchased from Laboratory Animal Research Center,Institute of Biophysics, Chinese Academy of Sciences. The mice werehoused under conditions of 4 mice/box, temperature 20 to 25° C., RH 40to 70% and 12 h:12 h day-night intermittently illumination. The micehave free access to food and drinking water, and the drinking water wasdistilled water prepared by the Laboratory Animal Research Center. Themice were divided into normal group, model group and groups of examples1 to 10, 8 mice per group.

2. Experimental Method

2.1 Test of Inhibition Rate of Tumor

Colon cancer cell line CT26 was subcutaneously inoculated to Balb/c miceat an inoculation dosage of 1×10⁶/mouse. 12 days later, the mice wereadministered by intragastric gavage with the compositions of examples 1to 10. The administration was performed once a day at a dosage of 500mg/kg/day. The normal group and the model group were administered byintragastric gavage with distilled water. On the 33^(th) day, 24 hoursafter the last administration, blood samples were collected from thehearts and the mice were sacrificed by cervical dislocation. The entiretumors were peeled off and weighed, and the inhibition rate of tumor wascalculated.The inhibition rate=(mean tumor weight of the model group−mean tumorweight of the administration group)/mean tumor weight of the modelgroup×100%.2.2 Test of CD4⁺ and CD8⁺ in Lymphocytes of Tumor Stroma andIntervention of the Traditional Chinese Medicine Composition

Tumors were taken from the mice by surgery under asepsis condition. Thetumors were weighted by an analytical balance and the tumors with aweight over 2 g were selected. A 1 mm³ block was constructed from theselected tumor tissues, disposed in a RPMI-1640 culture mediumcontaining 0.05% of collagenase, 0.02% of DNase, 0.01% of hyaluronidaseand 10% of calf serum, and subjected to digestion under magneticstirring at 4° C. overnight. The tissues were screened with a 200 meshessteel sieve. All the single cells were collected, subjected to low speedcentrifugation (1500 r/min) for 15 minutes, and washed with Hankssolution for twice. The cell concentration was adjusted to about 1×10⁶cells/mL, i.e., tumor infiltrating lymphocyte.

2 EP tubes were prepared for each test sample. 50 μL cell suspension wasadded to each tube and the total volume was adjusted to 100 μL with aPBS buffer to reach a final concentration of 5×10⁵ cells/mL. In onetube, 0.25 μg of anti-mouse CD4 monoclonal antibody and 0.2 μg ofanti-mouse CD8a monoclonal antibody labeled with different fluoresceinwere added, and the other tube was set as the blank control tube. Thetubes were incubated at room temperature for 30 minutes by avoidinglight. The cells were washed with PBS and tested by a flow cytometer.

2.3 Test of expression rate of VEGF and TGF-β1 positive cells in tumortissue Immunohistochemistry methods were used according to theprotocols. The results were determined by the appearance of brown colorin cytoplasm of the VEGF and TGF-β1 staining positive cells.2.4 Statistical Method

All the experimental data in the experiments was statistically analyzedwith software SPSS19.0. All the measurement data was expressed in theform of mean±standard deviation (−x±s), and t Test was performed. Andthe enumeration data was tested with χ2 Test. When P<0.05, thedifference is statistically significant.

3. Experimental Results

3.1 Tumor Inhibition Rate in Mice

TABLE 1 Inhibition effect of the samples on tumor growth in mice (n = 8)Group Normal Model Group Group Example 1 Example 2 Example 3 Example 4Tumor — — 31.36 35.27 32.39 33.20 Inhibition Rate (%) Group ExampleExample 5 Example 6 Example 7 Example 8 Example 9 10 Tumor 32.65 45.3740.16 39.82 42.81 41.12 Inhibition Rate (%)

As shown in Table 1, all the groups of examples 1 to 10 have inhibitioneffects on the growth of mice tumor. Therein, inhibition rate of Example6 was the highest, 45.37%; the second was Example 9, 42.81%; whileExample 1 has the least inhibition rate, 31.36%. The results indicatedthat both the individual components and the traditional Chinese medicinecompositions were capable to inhibit the growth of tumor and played arole in preventing and assisting in treating tumor. However, the effectsof the traditional Chinese medicine compositions were better than thatof the individual components.

3.2 Effects on CD4⁺, CD8⁺ Cells in Lymphocyte of Tumor Stroma.

TABLE 2 Effects of examples on CD4⁺, CD8⁺ cells in mice (n = 8) GroupCD4⁺(%) CD8⁺(%) Normal Group 45.82 ± 3.09 27.50 ± 1.21  Model Group 13.01 ± 1.13*   7.35 ± 0.69* Example 1 16.94 ± 1.79  8.35 ± 0.57Example 2  18.12 ± 2.47^(#)  9.19 ± 0.81 Example 3 17.24 ± 5.02  8.99 ±0.92 Example 4 17.46 ± 2.92  9.25 ± 0.47^(#) Example 5 17.46 ± 2.37 8.28 ± 1.14 Example 6  26.46 ± 2.37^(#) 14.28 ± 1.14^(#) Example 7 24.67 ± 2.11^(#) 13.09 ± 1.16^(#) Example 8 24.14 ± 6.22 12.78 ±2.07^(#) Example 9  25.09 ± 3.17^(#) 13.99 ± 1.45^(#) Example 10 24.75 ±5.32 12.95 ± 2.11^(#) Comment: comparing with the normal group, *P <0.05; and comparing with the model group, ^(#)P < 0.05.

As shown in Table 2, each of the groups of examples 1 to 10 increasedthe percentages of CD4⁺ and CD8⁺ cells in lymphocyte of tumor stroma insome degree. In addition, the CD4⁺ and CD8⁺ differences between examples6 to 10 and the model group were statistically significant (P<0.05).Therein, detection values of both CD4⁺ and CD8⁺ cells in the group ofExample 6 were the highest, (26.46±2.37)% and (14.28±1.14)%,respectively. The second was Example 9, with detection values of(25.09±3.17)% and (13.99±1.45)%, respectively. The results indicatedthat both the individual components and the traditional Chinese medicinecompositions increased the percentages of CD4⁺ and CD8⁺ cells andimproved the immune status of body, which could be used to producehealth products and foods for preventing and assisting in treatingtumor. However, the effects of the traditional Chinese medicinecompositions were better than that of the individual components.

3.3 Effects on Expression Rate of VEGF and TGF-β1 Positive Cells inTumor

TABLE 3 Effects of samples on VEGF and TGF-β1 positive cells in tumorGroup VEGF (%) TGF-β 1 (%) Normal Group 31.64 ± 3.09 35.76 ± 1.21 ModelGroup  75.43 ± 9.21*  70.36 ± 8.25* Example 1 69.28 ± 9.01 67.48 ± 7.92Example 2 63.79 ± 2.47 65.02 ± 0.81 Example 3 65.47 ± 5.02 66.41 ± 0.92Example 4  66.1 ± 2.92 66.73 ± 0.47 Example 5 65.31 ± 5.11 66.17 ± 0.83Example 6   58.49 ± 5.87 ^(#)   60.12 ± 3.28 ^(#) Example 7   60.99 ±2.11 ^(#)   61.12 ± 1.16 ^(#) Example 8 61.62 ± 6.22   61.63 ± 2.07 ^(#)Example 9   59.11 ± 3.17 ^(#)   60.78 ± 1.45 ^(#) Example 10 61.15 ±5.32   61.15 ± 2.11 ^(#) Comment: comparing with the normal group, *P <0.05; and comparing with the model group, ^(#)P < 0.05.

As shown in Table 3, each of the groups of examples 1 to 10 decreasedthe expression rate of VEGF and TGF-β1 positive cells in mice tumor.Therein, the differences of VEGF and TGF-β1 positive cells betweenexamples 6-10 and the model groups were statistically significant(P<0.05), while the differences between examples 1 to 5 and the modelgroup were not significant. Detection values of both the VEGF and TGF-β1positive cells of the group of Example 6 were the least, (58.49±5.87)%and (60.12±3.28)%, respectively. The second least was Example 9,(59.11±3.17)% and (60.78±1.45)%, respectively. The results indicatedthat both the individual components and the traditional Chinese medicinecompositions decreased the expression rate of VEGF and TGF-β1 positivecells in mice tumor, inhibited the expression of immune inhibitivefactors in local tumor tissue, therefore delaying the development oftumor and improving the immune status of body. They could be used inpreparing health products and foods, for preventing and assisting intreating tumor. However, the effects of the traditional Chinese medicinecompositions were better than that of the individual components.

It can be concluded from the above results that the traditional Chinesemedicine compositions comprising GANODERMA polysaccharide, MORINDAEOFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMA polysaccharide,ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil can inhibit thegrowth of tumors, improve percentages of CD4⁺ and CD8⁺ cells in thelymphocytes of the tumor stroma, and decrease the expression rate ofVEGF and TGF-β1 positive cells in mice tumor. The role was to inhibitthe growth of tumor cells, recover normal immune surveillance andprevent immune escape of the tumor cells. Therein, the traditionalChinese medicine composition which was prepared in Example 6 andconsisted of GANODERMA polysaccharide, MORINDAE OFFICINALIS RADIXpolysaccharide, POLYGONATI RHIZOMA polysaccharide, ANGELICAE SINENSISRADIX oil and MYRISTICAE SEMEN oil has the best effects on assisting intreating tumor and restoring the normal immune surveillance of body, andthe effects were better than that of the individual components (P<0.05).

Example 12 Compositions Comprising GANODERMA Polysaccharide, MORINDAEOFFICINALIS RADIX Polysaccharide, POLYGONATI RHIZOMA Polysaccharide,ANGELICAE SINENSIS RADIX Oil and MYRISTICAE SEMEN Oil with DifferentRatios, and Effects Thereof

GANODERMA polysaccharide prepared in Example 1, MORINDAE OFFICINALISRADIX polysaccharide prepared in Example 2, POLYGONATI RHIZOMApolysaccharide prepared in Example 3, ANGELICAE SINENSIS RADIX oilprepared in Example 4 and MYRISTICAE SEMEN oil prepared in Example 5were mixed according to the different weight ratios shown in the Table4. The compositions were subjected to the test methods of Example 11 forthe effects on preventing and/or assisting in treating tumors. Thescores of efficacy for restoring normal immune surveillance of the bodywere obtained (100 point scale, the efficacy for restoring normal immunesurveillance by the traditional Chinese medicine composition of Example6 with a weight ratio of 1:1:1.5:1.5:1.5 was recorded as 100 points, andthe percentages of other mixing ratios compared to the function testresult of Example 6 are recorded as the corresponding scores).

TABLE 4 Compositions comprising GANODERMA polysaccharide, MORINDAEOFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMA polysaccharide,ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil with differentweight ratios, and effects thereof Ratio of Components 1:5:5:1:51:5:4:2:4 4:2:5:1:1 2:1:1:2:1 1:2:2:1:1.5 1:1.5:1:1.5:1 1.5:1:1.5:1:1.5Score of Restoring 65 70 74 80 83 91 93 Normal Immune Surveillance

The results showed that comparing with using the individual componentsalone, the compositions comprising GANODERMA polysaccharide, MORINDAEOFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMA polysaccharide,ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil with a mixingratio of (1˜5):(1˜5):(1˜5):(1˜5):(1˜5) showed more significant effectson restoring normal immune surveillance of body. When the mixing ratiowas (1˜2):(1˜2):(1˜2):(1˜2):(1˜2), the effects were more significant.

Example 13 A Polysaccharide Composition Comprising GANODERMAPolysaccharide, MORINDAE OFFICINALIS RADIX Polysaccharide and POLYGONATIRHIZOMA Polysaccharide with a Ratio of 1:1:1.5, and Effects Thereof

GANODERMA polysaccharide prepared in Example 1, MORINDAE OFFICINALISRADIX polysaccharide prepared in Example 2 and POLYGONATI RHIZOMApolysaccharide prepared in Example 3 were mixed in the best weight ratioof 1:1:1.5. The composition was subjected to the test methods of Example11 for the effects on preventing and/or assisting in treating tumors.The scores of efficacy for restoring normal immune surveillance of thebody were obtained (100 point scale, the efficacy for restoring normalimmune surveillance by the traditional Chinese medicine composition ofExample 6 with a weight ratio of 1:1:1.5:1.5:1.5 was recorded as 100points, and the percentages of other mixing ratios compared to thefunction test result of Example 6 are recorded as the correspondingscores).

TABLE 5 Compositions comprising GANODERMA polysaccharide, MORINDAEOFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMA polysaccharide,ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil with differentratios, and effects thereof Ratio of Components 1:1:1.5:0:0 1:5:5:1:51:5:4:2:4 4:2:5:1:1 2:1:12:1 1:2:2:1:1.5 1:1.5:1:1.5:1 1.5:1:1.5:1:1.5Score of 47 65 70 74 80 83 91 93 Restoring Normal Immune Surveillance

The results showed that comparing with using the polysaccharidecomposition alone, the compositions comprising GANODERMA polysaccharide,MORINDAE OFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMApolysaccharide, ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oilwith a mixing ratio of (1˜5):(1˜5):(1˜5):(1˜5):(1˜5) showed moresignificant effects on restoring normal immune surveillance of body.

Example 14 A Composition Comprising ANGELICAE SINENSIS RADIX Oil andMYRISTICAE SEMEN Oil with a Ratio of 1:1, and Effects Thereof

ANGELICAE SINENSIS RADIX oil prepared in Example 4 and MYRISTICAE SEMENoil prepared in Example 5 were mixed in the best weight ratio of 1:1.The composition was subjected to the test methods of Example 11 for theeffects on preventing and/or assisting in treating tumors. The scores ofefficacy for restoring normal immune surveillance of the body wereobtained (100 point scale, the efficacy for restoring normal immunesurveillance by the traditional Chinese medicine composition of Example6 with a weight ratio of 1:1:1.5:1.5:1.5 was recorded as 100 points, andthe percentages of other mixing ratios compared to the function testresult of Example 6 are recorded as the corresponding scores).

TABLE 6 Compositions comprising GANODERMA polysaccharide, MORINDAEOFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMA polysaccharide,ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil with differentratios, and effects thereof Ratio of Components 0:0:0:1:1 1:5:5:1:51:5:4:2:4 4:2:5:1:1 2:1:1:2:1 1:2:2:1:1.5 1:1.5:1:1.5:1 1.5:1:1.5:1:1.5Score of Restoring 41 65 70 74 80 83 91 93 Normal Immune Surveillance

The results showed that comparing with using the oil composition alone,the compositions comprising GANODERMA polysaccharide, MORINDAEOFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMA polysaccharide,ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil with a mixingratio of (1˜5):(1˜5):(1˜5):(1˜5):(1˜5) showed more significant effectson restoring normal immune surveillance of body.

Example 15 Compositions Comprising GANODERMA Polysaccharide, MORINDAEOFFICINALIS RADIX Polysaccharide, POLYGONATI RHIZOMA Polysaccharide,ANGELICAE SINENSIS RADIX Oil and MYRISTICAE SEMEN Oil with DifferentRatios, and Effects Thereof

GANODERMA polysaccharide prepared in Example 1, MORINDAE OFFICINALISRADIX polysaccharide prepared in Example 2, POLYGONATI RHIZOMApolysaccharide prepared in Example 3, ANGELICAE SINENSIS RADIX oilprepared in Example 4 and MYRISTICAE SEMEN oil prepared in Example 5were mixed according to the weight ratios shown in Table 7 (eachcomposition lacked one or two components). The compositions weresubjected to the test methods of Example 11 for the effects onpreventing and/or assisting in treating tumors. The scores of efficacyfor restoring normal immune surveillance of the body were obtained (100point scale, the efficacy for restoring normal immune surveillance bythe traditional Chinese medicine composition of Example 6 with a weightratio of 1:1:1.5:1.5:1.5 was recorded as 100 points, and the percentagesof other mixing ratios compared to the function test result of Example 6are recorded as the corresponding scores).

TABLE 7 Compositions comprising GANODERMA polysaccharide, MORINDAEOFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMA polysaccharide,ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oil with differentratios, and effects thereof Ratio of Components 1:5:5:1:5 1:5:4:2:44:2:5:1:1 2:1:1:2:1 1:2:2:1:1.5 1:1.5:1:1.5:1 1.5:1:1.5:1:1.5 Score of65 70 74 80 83 91 93 Restoring Normal Immune Surveillance Ratio ofComponents 1:1:1.5:0:1.5 1:0:0:1.5:1.5 0:0:1:1:0 1:1:1.5:1.5:0 0:0:1:1:10:1:0:1.5:1.5 0:1:0:0:1.5 Score of 61 60 56 58 53 60 54 Restoring NormalImmune Surveillance

The results showed that comparing with the compositions that lacked oneor two components, the compositions comprising GANODERMA polysaccharide,MORINDAE OFFICINALIS RADIX polysaccharide, POLYGONATI RHIZOMApolysaccharide, ANGELICAE SINENSIS RADIX oil and MYRISTICAE SEMEN oilwith a mixing ratio of (1˜5):(1˜5):(1˜5):(1˜5):(1˜5) showed moresignificant effects on restoring normal immune surveillance of body.When the mixing ratio was (1˜2):(1˜2):(1˜2):(1˜2):(1˜2), the effectswere more significant.

What is claimed is:
 1. A method of inhibiting the growth of tumor,increasing the percentage of CD₄ ⁺ and CD₈ ⁺ cells in lymphocytes oftumor stroma, decreasing VEGF and TGF-β1 positive cells in tumor tissue,enhancing immunity, preventing and/or assisting in treating tumor,and/or restoring normal immune function, comprising administrating aneffective amount of a traditional Chinese medicine composition to asubject in need thereof; wherein the traditional Chinese medicinecomposition comprises GANODERMA polysaccharide, MORINDAE OFFICINALISRADIX polysaccharide, POLYGONATI RHIZOMA polysaccharide, ANGELICAESINENSIS RADIX oil and MYRISTICAE SEMEN oil, wherein the weight ratio ofGANODERMA polysaccharide, MORINDAE OFFICINALIS RADIX polysaccharide,POLYGONATI RHIZOMA polysaccharide, ANGELICAE SINENSIS RADIX oil andMYRISTICAE SEMEN oil is (1 to 5):(1 to 5):(1 to 5):(1 to 5):(1 to 5). 2.The method according to claim 1, wherein the weight ratio of GANODERMApolysaccharide, MORINDAE OFFICINALIS RADIX polysaccharide, POLYGONATIRHIZOMA polysaccharide, ANGELICAE SINENSIS RADIX oil and MYRISTICAESEMEN oil is (1 to 2):(1 to 2):(1 to 2):(1 to 2):(1 to 2).
 3. The methodaccording to claim 1, wherein the weight ratio of GANODERMApolysaccharide, MORINDAE OFFICINALIS RADIX polysaccharide, POLYGONATIRHIZOMA polysaccharide, ANGELICAE SINENSIS RADIX oil and MYRISTICAESEMEN oil is 1:1:1.5:1.5:1.5.
 4. The method according to claim 1, wherein the traditional Chinese medicine composition is in the form of ahealth food or a medicine.
 5. The method according to claim 4, whereinthe health food or the medicine further comprises a pharmaceuticallyacceptable excipient or a food acceptable excipient.